Abstract The life-threatening diarrheal disease cholera is caused by toxigenic strains of the Gram-negative organism Vibrio cholerae. In addition to the well-characterized ADP-ribosylating cholera toxin (CT), V. cholerae secretes a novel toxin that is the founding member of new family of Multifunctional, Autoprocossin RTX toxins (MARTX). The MARTX toxin of V. cholerae (MARTX-Vc) contributes to virulence in mice and may be an important factor contributing to persistent colonization both in cholera patients and non-symptomatic carriers.. MARTX-Vc is of intrinsic interest due to its novel biochemical properties and mode of action. At a predicted size of >480 kDa, the toxin is a multifunctional toxin. Thus far, we have described three activities associated with this toxin. We have demonstrated that this toxin depolymerizes actin stress fibers and covalently crosslinks actin into oligomers. We have demonstrated that this toxin causes rounding of cells due to inactivation of RhoGTPases by an unknown mechanism. Finally, this toxin has a cysteine protease domain with autoprocessing activity that may also have cellular targets resulting in cytotoxicity. Each of these activities has been mapped to a specific domain of the toxin. A fourth domain homologous to the alpha-beta hydrolase family of proteins is also a putative activity domain. In this grant proposal, we will pursue the multiple catalytic activities of the toxin by studying the mechanism of action of the independent activity domains. A structure [unreadable] function analysis of the actin crosslinking domain will be performed and the regions essential for catalysis and actin binding will be identified. The target of the RhoGTPase inactivating domain will be identified and characterized. The requirements for autocleavage site recognition and inositol hexakisphosphate binding will be investigated and other potential sites of cleavage identified. In all, this work will further our understanding of the mechanism of action of this toxin and provide insight into its role in disease and will also provide information about related toxins that share the novel functional domains carried by this unique multifunctional toxin. Lay summary The life-threatening diarrheal disease cholera is caused the Gram-negative organism Vibrio cholerae. In addition to the well-characterized ADP-ribosylating cholera toxin (CT), V. cholerae secretes a novel toxin that is the founding member of new family of the RTX toxins, the Multifunctional-Autoprocessing RTX toxins (MARTX). The MARTX toxin of V. cholerae contributes to virulence in mice and may be an important factor contributing to persistent colonization both in cholera patients and non-symptomatic carriers. This proposal seeks to understand the cellular mechanism of action of this toxin that has three known enzymatic activities: actin crosslinking, Rho-inactivation, and autoprocessing. Completion of this research will impact not only our understanding of cholera pathogenesis, but also the function of other uncharacterized toxins produced by other human pathogens that share these unique enzymatic activities.